Department of Integrative Biology and Physiology, University of California, Los Angeles, CA, USA.
Methods Mol Biol. 2024;2782:123-136. doi: 10.1007/978-1-0716-3754-8_9.
The role of immune system in the progression of neurodegenerative diseases has been studied for decades in animal models. However, invasive studies in human subjects remain controversial due to the heterogeneity of the presentation of different diagnostic categories at different stages of the disease. Peripheral blood mononuclear cells (PBMCs) contain immune cells including dendritic cells (DCs), monocytes, macrophages, and T lymphocytes. Isolating PBMCs from whole blood samples collected from patients provides a minimally invasive method for analyzing the immune system's function in patients with neurodegenerative diseases. By isolating single cell types from patients' peripheral blood, in vitro analyses can be conducted including RNA sequencing, immunofluorescence, and phagocytic analysis. In this chapter, we discuss PBMC separation and isolation of macrophages in pure culture in vitro. We also outline methods for performing RNA-seq on cultured macrophages and other techniques for investigating the role of macrophages in neurodegenerative disease pathophysiology.
免疫系统在神经退行性疾病进展中的作用已在动物模型中研究了数十年。然而，由于不同诊断类别在疾病不同阶段的表现存在异质性，对人类受试者进行侵入性研究仍存在争议。外周血单核细胞（PBMCs）包含免疫细胞，包括树突状细胞（DCs）、单核细胞、巨噬细胞和 T 淋巴细胞。从从患者采集的全血样本中分离 PBMCs 提供了一种微创方法，可用于分析神经退行性疾病患者免疫系统的功能。通过从患者外周血中分离单个细胞类型，可以进行体外分析，包括 RNA 测序、免疫荧光和吞噬分析。在本章中，我们讨论了 PBMC 分离和体外纯培养巨噬细胞的分离。我们还概述了在培养的巨噬细胞上进行 RNA-seq 的方法以及其他用于研究巨噬细胞在神经退行性疾病发病机制中的作用的技术。
